Phosphorylation of DARPP-32 regulates breast cancer cell migration downstream of the receptor tyrosine kinase DDR1

Abstract

Cell migration plays a central role in processes such as development, wound healing and cancer metastasis. Here we describe a novel interaction between DDR1, a receptor tyrosine kinase activated by collagen, and the phosphoprotein DARPP-32 in mammary epithelial cells. DARPP-32 expression was readily detected in non-transformed mammary cell lines, but was strongly reduced or even absent in breast tumor cell lines, such as MCF7. Transfection of MCF7 cells with DARPP-32 resulted in severely impaired cell migration, while DARPP-32 transfection into the DDR1-deficient breast cancer cell line MDA-MB-231 did not alter migration. Co-expression of both DDR1 and DARPP-32 in MDA-MB-231 cells inhibited migration, thereby supporting a critical role of the DDR1/DARPP-32 complex in motility. Mutational substitution of the phosphorylation sites Thr-34 or Thr-75 on DARPP-32 revealed that phosphorylation of Thr-34 is necessary for the ability of DARPP-32 to impair breast tumor cell migration. Thus, DARPP-32 signaling downstream of DDR1 is a potential new target for effective anti-metastatic breast cancer therapy.

Introduction

Breast cancer is the most common form of cancer in women. While the prognosis for breast cancer patients without local or distal dissemination is relatively favorable, the prognosis is considerably worse once distal metastasis has been established. It is therefore imperative to identify molecular targets and develop novel anti-metastatic therapies that will stop, reduce, or delay the dissemination and growth of breast cancer metastasis. In this context, it is important to note that normal breast development displays many molecular events also found in breast cancer progression. Consequently, it is not surprising that proteins critical for mammary gland organogenesis are also found to promote or impair breast cancer growth or dissemination [1].

Discoidin Domain Receptor 1 (DDR1) is a receptor tyrosine kinase (RTK) widely expressed in epithelial cells of the mammary gland, lung, colon and pancreas [2], [3], [4], [5]. DDR1 plays a crucial role in mammary gland development since knockout mice show aberrant branching of the mammary ducts, poorly differentiated epithelium and fail to lactate at parturition [6], [7]. Increased DDR1 mRNA expression was found in breast, lung, ovarian and esophageal cancer, and elevated DDR1 protein levels confirmed in the two latter types of cancer [4], [8]. Although the available clinical data have failed to yet show a correlation between increased DDR1 protein expression and breast cancer prognosis, it is possible that a correlation between the activity of DDR1 and cancer prognosis exists since somatic mutations were recently identified in lung cancer samples [9], [10].

The ligand for DDR1 is native collagen, and collagen type I–V and VIII were found to be equally effective [11]. Compared to other RTKs, the activation of DDR1 is unusually slow, requiring several hours to reach maximal activation. Experiments performed in a variety of cell types, including vascular smooth muscle cells, epithelial cells and monocytes, have provided evidence that DDR1 is critically involved in regulating cell adhesion and migration [12], [13], [14], [15]. Upon activation of its catalytic function, a number of cytoplasmic signaling molecules, such as ShcA, FRS-1, Nck2 and Shp-2, bind DDR1 in a phosphotyrosine-dependent manner [11], [16], [17], [18].

Dopamine and cAMP-regulated neuronal phosphoprotein, molecular mass 32 kDa, (DARPP-32) was first identified in brain regions enriched in dopaminergic nerve terminals more than two decades ago [19]. Since then, a large body of work has shown that DARPP-32 is a central signaling molecule activated by a diverse array of neurotransmitters such as dopamine, glutamate, serotonin and GABA [20]. Using knockout mice, it was found that DARPP-32 triggers dopamine receptor evoked signaling leading to activation of mitogen-activated protein kinases, CREB and c-fos [21]. Upon neurotransmitter stimulation, DARPP-32 is phosphorylated at multiple sites and can function both as a kinase inhibitor and as a phosphatase inhibitor [20]. Specifically, phosphorylation of DARPP-32 on threonine at position 34 (Thr-34) by protein kinase A (PKA) converts it into a potent inhibitor of protein phosphatase 1 (PP1), while phosphorylation on Thr-75 by Cdk5 or Cdc2 inhibits PKA action [22], [23]. Aside from the extensive research in the brain, a potential function of DARPP-32 outside of the nervous system has only very recently been explored. Messenger RNA expression and immunohistochemistry analysis revealed that DARPP-32 is overexpressed in gastric cancers [24], [25]. Subsequent work showed expression in a number of adenocarcinomas such as colon, prostate and esophageal cancers, suggesting at least a supportive function in oncogenesis [26]. However, the role of DARPP-32 in different cancer entities is mostly likely complex since a recent survey of primary esophageal cancer samples reported that patients expressing DARPP-32 have a better prognosis than those who are DARPP-32 negative [27]. Clearly, more research is required to resolve these apparently opposing findings.

Here we have identified DARPP-32 as a novel binding partner of DDR1 in human mammary cells and shown that DARPP-32 protein expression is either reduced or lost in various breast cancer cell lines. Furthermore, we find that re-expression of DARPP-32 impairs migration of breast tumor cells via a DDR1- and Thr-34 phosphorylation-dependent mechanism.